RESUMO
Fracture healing is a complex process with multiple overlapping metabolic and differentiation phases. Small non-coding RNAs are involved in the regulation of fracture healing and their presence in circulation is under current interest due to their obvious value as potential biomarkers. Circulating microRNAs (miRNAs) have been characterized to some extent but the current knowledge on tRNA-derived small RNA fragments (tsRNAs) is relatively scarce, especially in circulation. In this study, the spectrum of circulating miRNAs and tsRNAs was analysed by next generation sequencing to show their differential expression during fracture healing in vivo. Analysed tsRNA fragments included stress-induced translation interfering tRNA fragments (tiRNAs or tRNA halves) and internal tRNA fragments (i-tRF), within the size range of 28-36 bp. To unveil the expression of these non-coding RNAs, genome-wide analysis was performed on two months old C57BL/6 mice on days 1, 5, 7, 10, and 14 (D1, D5, D7, D10, and D14) after a closed tibial fracture. Valine isoacceptor tRNA-derived Val-AAC 5'end and Val-CAC 5'end fragments were the major types of 5'end tiRNAs in circulation, comprising about 65 % of the total counts. Their expression was not affected by fracture. After a fracture, the levels of two 5'end tiRNAs Lys-TTT 5' and Lys-CTT 5' were decreased and His-GTG 5' was increased through D1-D14. The level of miR-451a was decreased on the first post-fracture day (D1), whereas miR-328-3p, miR-133a-3p, miR-375-3p, miR-423-5p, and miR-150-5p were increased post-fracture. These data provide evidence on how fracture healing could provoke systemic metabolic effects and further pinpoint the potential of small non-coding RNAs as biomarkers for tissue regeneration.
RESUMO
Long-bone fracture is a common injury and its healing process at the fracture site involves several overlapping phases, including inflammation, migration of mesenchymal progenitors into the fracture site, endochondral ossification, angiogenesis and finally bone remodelling. Increasing evidence shows that small noncoding RNAs are important regulators of chondrogenesis, osteogenesis and fracture healing. MicroRNAs are small single-stranded, non-coding RNA-molecules intervening in most physiological and biological processes, including fracture healing. Angiogenin-cleaved 5' tRNA halves, also called as tiRNAs (stress-induced RNAs) have been shown to repress protein translation. In order to gain further understanding on the role of small noncoding RNAs in fracture healing, genome wide expression profiles of tiRNAs, miRNAs and mRNAs were followed up to 14 days after fracture in callus tissue of an in vivo mouse model with closed tibial fracture and, compared to intact bone and articular cartilage at 2 months of age. Total tiRNA expression level in cartilage was only approximately one third of that observed in control D0 bone. In callus tissue, 11 mature 5'end tiRNAs out of 191 tiRNAs were highly expressed, and seven of them were differentially expressed during fracture healing. When comparing the control tissues, 25 miRNAs characteristic to bone and 29 miRNAs characteristic to cartilage tissue homeostasis were identified. Further, a total of 54 out of 806 miRNAs and 5420 out of 18,700 mRNAs were differentially expressed (DE) in callus tissue during fracture healing and, in comparison to control bone. They were associated to gene ontology processes related to mesenchymal tissue development and differentiation. A total of 581 miRNA-mRNA interactions were identified for these 54 DE miRNAs by literature searches in PubMed, thereby linking by Spearman correlation analysis 14 downregulated and 28 upregulated miRNAs to 164 negatively correlating and 168 positively correlating miRNA-mRNA pairs with chondrogenic and osteogenic phases of fracture healing. These data indicated that tiRNAs and miRNAs were differentially expressed in fracture callus tissue, suggesting them important physiological functions during fracture healing. Hence, the data provided by this study may contribute to future clinical applications, such as potential use as biomarkers or as tools in the development of novel therapeutic approaches for fracture healing.